Many chemopreventive agents activate the Nrf2 transcription factor, leading to upregulation of cytoprotective enzymes and Nrf2-dependent protection against tumorigenesis. Under basal conditions, Nrf2 is sequestered in the cytoplasm and targeted for ubiquitination and degradation by Keap1. Oxidative stress or chemopreventive agents lead to Nrf2 escape from Keap1 repression, allowing Nrf2 to upregulate cytoprotective genes by binding to their antioxidant response elements (AREs). The specific goals of this project are to investigate the role of Nrf2 Ser40 phosphorylation in this mechanism for chemopreventive agents, to challenge an existing assumption that downregulation of ubiquitination is sufficient for maximum ARE activation, and to determine whether Ser40 phosphorylation alters the cytoplasmic/nuclear distribution of Nrf2, leading to ARE activation. The broad objectives of this project are to determine the mechanisms by which chemopreventive agents upregulate cytoprotective enzyme expression by the ARE, and compare these to those utilized by oxidative stress inducing agents. It is anticipated that this information will ultimately be used to develop a method to distinguish between these two types of agents that function via the Nrf2 pathway, in order to aid development of agents that are primarily cytoprotective. This information is also anticipated to be valuable in determining a target in this pathway for drug screening and design. To accomplish the goals of this project, we propose the following specific aims: Specific Aim 1. Determine whether Nrf2 Ser40 is phosphorylated in response to a series of inducers, including promising chemopreventive agents, and whether it is required for Nrf2 nuclear accumulation. Tagged Nrf2 will be extracted from cells treated with inducers, and a mass spectrometry technique termed AQUA will be used to determine to what extent serine 40 is phosphorylated in response to inducers. In addition, cells transfected with Nrf2 S40A will be evaluated for ARE activation in response to the series of inducers, with wt Nrf2 as a control. Specific Aim 2. Show that downregulation of Nrf2 ubiquitination and subsequent Nrf2 stabilization in response to chemopreventive agents are in fact insufficient for ARE activation, and that phosphorylation of Ser40 leads to ARE activation by inducing nuclear accumulation of Nrf2. These experiments will be conducted in tissue culture. The dependence of ARE activation on Nrf2 ubiquitination will be determined via a two-pronged approach using a proteasome inhibitor and an Nrf2 mutant that has the ubiquitination sites mutated to Arg. Phosphorylation at Ser40 will be monitored using AQUA, or the Nrf2 S40A and S40E mutants will be used. ARE activation will be measured using a luciferase assay. Cellular levels, as well as the ubiquitination status, of the mutant and wild type Nrf2 proteins will be evaluated by immunoblotting, and Nrf2 localization will be determined by probing nuclear/cytoplasmic extracts. Keap1 levels and ubiquitination will be monitored as well.